Advancing structural elucidation of conjugation drug metabolites in metabolite profiling with novel electron-activated dissociation.

ABSTRACT

RATIONALE This study focuses on the advantage of using the novel electron-activated dissociation (EAD) technology on the QTOF system for structural elucidation of conjugation metabolites. In drug metabolite identification, conceptual "boxes" are generally used to represent potential sites of modifications, which are proposed based on MS/MS data. Electron-activated dissociation (EAD) provides unique fragmentation patterns, potentially allowing for more precise localization of the metabolic modification sites compared to CID, particularly for conjugations. METHOD: Known compounds were incubated with rat liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), uridine dihosphate-glucuronic acid (UDPGA), and glutathione. Conjugation metabolites were analyzed using the QTOF system. High-resolution MS/MS spectra were collected using EAD and CID fragmentations along with TOF MS full scan for tested drugs and metabolites. Fragmentation patterns were compared to evaluate their efficiency in structural elucidation. RESULTS: Metabolite profiling identified conjugation metabolites (glucuronides and GSH adducts), using characteristic mass shifts. A comparison of EAD and CID fragmentation revealed EAD-specific fragments for most conjugates. EAD was able to break the relatively stable bonds on parent drug motifs while keeping relatively weak conjugation bonds intact, despite the generally low intensity of EAD. EAD effectively narrowed the conceptual "box" representing modification sites, providing more definitive information on conjugation sites and facilitating the structural elucidation of conjugated metabolites. CONCLUSION: EAD is a powerful tool for metabolite profiling in drug development, particularly for identifying conjugation sites. EAD-enabled MS/MS spectra offer a greater variety of signature fragments compared to CID, resulting in more comprehensive and unique structural information for metabolic modification analysis. Overall, EAD, complementary to CID, has the potential to narrow down potential modification sites, significantly enhancing the precision of conjugation metabolite structure elucidation.